Abstract : Conventional techniques for single-base resolution mapping of epigenetic modifications of DNA such as 5-hydroxymethylcytosine 5hmC rely on the sequencing of bisulfite-modified DNA. Here we present an alternative approach called SCL-exo which combines selective chemical labeling SCL of 5hmC in genomic DNA with exonuclease exo digestion of the bead-trapped modified DNA molecules. Associated with a straightforward bioinformatic analysis, this new procedure provides an unbiased and fast method for mapping this epigenetic mark at high resolution. Implemented on mouse genomic DNA from in vitro-differentiated neural precursor cells, SCL-exo sheds light on an intrinsic lack of conservation of hydroxymethylated CpGs across vertebrates.